Method for the in vitro synthesis of short double stranded rnas

ABSTRACT

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

[0001] The present invention relates to the field of synthesis of shortdouble-stranded target-specific RNAs. An in vitro transcription methodusing RNA polymerases and target sequence-specific DNA oligonucleotidesas templates is disclosed. The present invention finds particularlyadvantageous use in the synthesis of short interfering RNAs (siRNAs)that have been shown to function as key intermediates in triggeringsequence-specific RNA degradation during posttranscriptional genesilencing in plants and RNA interference in invertebrates and vertebratesystems.

BACKGROUND OF THE INVENTION

[0002] RNA silencing is a remarkable type of gene regulation based onsequence-specific targeting and degradation of RNA. RNA silencing wasfirst discovered in transgenic plants, where it was termed cosuppressionor posttranscriptional gene silencing (PTGS). Only recently asequence-specific RNA degradation process, RNA interference (RNAi),related to PTGS has been found in ciliates, fungi and a variety ofanimals from C. elegans to mice and human cells. Although they maydiffer in detail, RNAi and PTGS result from the same highly conservedmechanism, indicating an ancient origin. The basic process involves adouble stranded RNA (dsRNA) that is cleaved into small double strandedinterfering RNAs (siRNA) which guide recognition and targeted cleavageof homologous mRNA. These small dsRNAs resemble breakdown products of anRNase III-like digestion. In particular, siRNAs are target-specificshort double stranded RNAs wherein each strand of the siRNAs carries5′monophosphate, 3′hydroxyl termini and 3′ overhangs of 2-3 nucleotides(Caplen, N. et al., 2001, PNAS (98) 9742-9747).

[0003] RNAi has attracted considerable attention because it is a meansof knocking out the activity of specific genes, being particularlyuseful in species that were previously considered not to be amendable togenetic analysis. Recent studies demonstrated that synthetic siRNAs caninduce gene-specific inhibition of expression in C. elegans and in celllines from humans and mice (Caplen N., et al., 2001, PNAS (98)9742-9747; Elbashir S., et al., 2001, Nature (411) 494-498). In saidpublications it was further demonstrated that in mammalian cells siRNAsprovide a sequence specific answer compared to the use of longer dsRNAswhich inactivate the translation factor eIF2α, leading to a generalizedsuppression of protein synthesis. Also, in comparison to inhibition ofgene expression using antisense technology, siRNAs seem to be verystable and thus may not require the extensive chemical modificationsthat single stranded RNA antisense oligonucleotides require to enhancethe in vivo half life.

[0004] It is therefore to be expected that RNA silencing using siRNAswill become an important tool in engineering control of gene expressionas well as in functional genomics and a variety of biotechnologyapplications ranging from molecular farming to possibly even genetherapy in animals. As different siRNAs may work with differenteffectiveness on their targets, the testing of more than one siRNA for aparticular target will be desirable. In addition, genome-scale reversegenetics programs will require large numbers of siRNAs.

[0005] However, production of double stranded target-specific RNA oligosby traditional chemical synthesis remains relatively slow and expensivewhen compared to DNA oligo synthesis. In addition, chemical synthesis ofRNA oligos requires special synthesizers and complex purificationprotocols. The present invention provides an alternative approach toproduce short double stranded target-specific RNAs based on in vitrotranscription using bacteriophage or other viral polymerases and targetsequence-specific oligonucleotide templates. Compared to the chemicalsynthesis of RNA oligos the present invention is relatively quick andeasy to perform.

[0006] However, the in vitro transcribed siRNAs differ from thechemically synthesized RNA oligos in two ways. Primarily, identical tothe natural occurring siRNAs, the chemically synthesized RNA oligos havea 5′monophosphate group. The in vitro transcribed siRNAs retain a5′triphosphate group. It was unknown whether the presence of thistriphosphate group renders the in vitro transcribed siRNAs incompetentto induce RNA interference.

[0007] Secondly, chemically synthesized RNA oligos are highly purifiedusing amongst others Ion Exchange and Reverse Phase HPLC wherein purityand quality of the synthesized compounds is further evaluated usingamongst others NMR and mass spectrometry analysis. In the presentinvention a simple, crude purification protocol is used comprising sizeexclusion chromatography, phenol:chloroform extraction and ethanolprecipitation. It was again uncertain whether the ommitance of anextensive purification protocol would affect the usefulness of “invitro” transcribed RNAs in RNA-mediated silencing.

[0008] Surprisingly, the present invention demonstrates that the5′triphosphate group and the crude purification does not affect the RNAsilencing activity of “in vitro” transcribed RNAs and provides analternative approach to siRNA synthesis which makes it accessible as aresearch tool in an average molecular biology laboratory.

[0009] Existing in vitro methods to synthesize small single strandedRNAs of defined length and sequence (Milligan F. et al., 1987, NucleicAcid Res. (15) 8783-8798), were not directly applicable for thesynthesis of small interfering RNAs. The problem resides in the factthat RNA polymerases tend to transcribe some nucleotides from thepromoter sequence into the transcript. As a consequence, thetarget-specific dsRNAs which can be produced by annealing complementarysingle stranded RNA molecules generated using the aforementionedmethods, must comprise at the 5′end the nucleotides transcribed from thepromoter sequence and at the 3′end the nucleotides complementary to thenucleotides transcribed from the promoter sequence. It may well be thatin the mRNA of the target sequence no stretch of a defined sequencelength exists wherein the 5′-end consists of the nucleotides transcribedfrom the promoter sequence and the 3′-end of the nucleotidescomplementary to the nucleotides transcribed from the promoter sequence.The present invention solves this problem by providing truncated RNApolymerase promoter sequences wherein one or more nucleotides at the5′end of the template strand of the promoter sequence are replaced bynucleotides that are part of the target-specific sequence. Thesesubstitutions do not affect the in vitro transcription yields, butincrease the possibility that at least one target-specific sequence of adefined sequence length exists in the mRNA of the target protein,wherein the 5′-end consists of the nucleotides transcribed from thepromoter sequence and the 3′-end of the nucleotides complementary to thenucleotides transcribed from the promoter sequence.

[0010] This and other aspects of the invention will be described hereinbelow.

SUMMARY OF THE INVENTION

[0011] The present invention provides an in vitro method for thesynthesis of short double stranded target-specific RNAs comprising thesteps of a) combining a sense target-specific oligonucleotide templateand a chain extending enzyme in a reaction mixture such that thetemplate extended sense oligoribonucleotide product is formed; b)combining an antisense target-specific oligonucleotide template and achain extending enzyme in a reaction mixture such that the templateextended antisense oligoribonucleotide product is formed; and c)hybridizing the sense oligoribonucleotide product obtained in step a)with the complementary antisense oligoribonucleotide product obtained instep b).

[0012] In a further embodiment of the present invention the chainextending enzyme is an RNA polymerase and the oligonucleotide templatesof step a) and b) comprise an RNA polymerase promoter sequence,preferably consisting of dsDNA. In a more preferred embodiment the RNApolymerase is T7 polymerase and the oligonucleotide templates of step a)and b) comprise a T7 RNA polymerase promoter sequence extended at the5′end of the template strand with the target-specific template sequence,optionally extended with 2 or 3 additional nucleotides. The presentinvention finds particular use in the synthesis of small interferingRNAs. It is therefore, a further objective of the present invention toprovide a method for the synthesis of target-specific short doublestranded RNAs, wherein said target-specific short double stranded RNAsare less than 50 nucleotides, preferably less than 30 nucleotides long,even more preferably 30-12 nucleotides long, further characterised bycomprising at the 5′-end nucleotides transcribed from the promotersequence and at the 3′-end nucleotides complementary to the nucleotidestranscribed from the promoter sequence.

[0013] Accordingly, the present invention provides a method for thesynthesis of small interfering RNAs comprising the steps of a) combininga sense siRNA template with a chain extending enzyme in a reactionmixture such that the template extended sense oligoribonucleotideproduct is formed; b) combining an antisense siRNA template with a chainextending enzyme in a reaction mixture such that the template extendedantisense oligoribonucleotide product is formed; and c) hybridizing thesense oligoribonucleotide product obtained in step a) with the antisenseoligoribonucleotide product obtained in step b); wherein the siRNAtemplates of step a) and b) comprise a double stranded RNA polymerasepromoter sequence extended at the 5′end of the template strand with thetarget-specific template sequence and 2 or 3 additional nucleotides. Ina preferred embodiment the chain extending enzyme is T7 RNA polymeraseand the siRNA templates comprise a double stranded T7 RNA polymerasepromoter sequence, preferably the truncated T7 RNA polymerase promotersequence shown in FIG. 1.

[0014] It is a further object of the present invention to provide kitsto perform the methods according to the invention as well as thecompounds for use in any of the methods disclosed.

BRIEF DESCRIPTION OF THE DRAWING

[0015]FIG. 1: Oligonucleotide production scheme. An example is given forthe design of siRNA oligonucleotide templates for a target sequence of19 nucleotides within the coding sequence of JNK2α1 mRNA.

[0016]FIG. 2A: GL3 target-specific double stranded siRNAs used in aluciferase reporter assay. GL3 siRNA 1, GL3 siRNA 2 and GL3 siRNA 3 weremade using the in vitro method of the invention. GL3 siRNA oligo waschemically synthesized (Vargeese, C. et al., 1988, Nucleic Acid Res.(26), 1046-1050)

[0017]FIG. 2B: Effects of GL3 target-specific siRNAs and of GL3antisense single stranded siRNAs on luciferase expression in HeLa cells.Cells transfected with GL3-control luciferase+reporter constructs weretaken as 100%.

[0018]FIG. 2C: Dose response curve of the GL3-siRNA 1 inhibitory effecton luciferase expression in pGL3-control transfected HeLa cells..

[0019]FIG. 3A: EGFP target-specific double stranded siRNAs used in aFACS analysis of EGFP-transfected HeLa cells. EGFP ds siRNA 2 was madeusing the in vitro method of the invention. EGFP ds siRNA oligo waschemically synthesized (Vargeese, C. et al., 1988, Nucleic Acid Res.(26), 1046-1050)

[0020]FIG. 3B: Effects of EGFP target-specific siRNAs and of EGFPantisense single stranded siRNAs on GFP fluorescence in EGFP-transfectedHeLa cells using FACScan analysis (Beckton-Dickinson). Cells transfectedwith EGFP DNA only were taken as 100%.

[0021]FIG. 4A: JNK2α1 target-specific double stranded siRNAs used inJNK2α1 transfected HeLa cells.

[0022]FIG. 4B: CDS-1 target-specific double stranded siRNAs used inCDS-1 transfected HeLa cells.

DETAILED DESCRIPTION

[0023] This invention relates to the field of synthesis of shortdouble-stranded target-specific RNAs and is based on the in vitrotranscription of oligonucleotide templates using chain extendingenzymes.

[0024] Target-specific short double stranded RNAs as used herein refersto a double-stranded RNA that matches part of the sequence encoding fora specific protein, i.e. the target protein. These sequences arepreferably less than 50 nucleotides, more preferably less than 30nucleotides long, even more preferably 15-25 nucleotides long. In aparticular embodiment the target-specific short double stranded RNAs areuseful in RNA interference in invertebrate and invertebrate systems assmall interfering RNAs (siRNAs). RNAs as used herein are short dsRNAmolecules of 12-30 nucleotides, with 2- or 3-nucleotide overhanging3′-ends. In a preferred embodiment the siRNAs are 15-25 nucleotides longwith 2 nucleotide overhanging 3′ends. Even more preferred the siRNAs are17-22 nucleotides long with 2 nucleotide overhanging 3′ends.

[0025] In order to obtain dsRNA both a sense and an antisenseoligonucleotide template are required. The term “oligonucleotidetemplates” as used herein refers to structures that in some directphysical process can cause the patterning of a second structure, usuallycomplementary to it in some sense. In current biology almost exclusivelyused to refer to a nucleotide sequence that directs the synthesis of asequence complementary to it by the rules of Watson-Crick base-pairing(The Dictionary of Cell and Molecular Biology, 3d. Edition, AcademicPress, London, 1999 (ISBN 0-12-432565-3)). These template sequences arepreferably less than 50 nucleotides long, and may either be doublestranded, single stranded or partially single stranded DNA oligotemplates.

[0026] The oligonucleotide templates could either be synthetic DNAtemplates or templates generated as linearized plasmid DNA from atarget-specific sequence cloned into a restriction site of a vector suchas for example a prokaryotic cloning vector (pUC13, pUC19) or PCRcloning systems such as the TOPO cloning system of Invitrogen. Thesynthetic DNA templates may be produced according to techniques wellknown in the art. In a preferred embodiment of the present invention theoligonucleotide templates consist of partially single-stranded DNA oligotemplates comprising an RNA polymerase promoter sequence consisting ofdsDNA. In this embodiment the target-specific short double stranded RNAsare further characterized by comprising at the 5′end nucleotidestranscribed from the RNA polymerase promoter sequence and at the 3′endnucleotides complementary to the nucleotides transcribed from thepromoter sequence.

[0027] A “Chain extending enzyme” as defined herein refers to an enzymecapable of forming an RNA polymer from ribonucleoside 5′triphosphates;the RNA formed is complementary to the DNA template. The enzyme addsmononucleotide units to the 3′-hydroxyl ends of the RNA chain and thusbuilds RNA in the 5′→3′ direction, antiparallel to the DNA strand usedas template. Such chain extending enzymes could for example be DNAdependent polymerases such as DNA polymerase I, II and III; RNA-directedDNA polymerases such as RSV and AMV-polymerases; DNA-directed RNApolymerases such as E.coli RNA polymerase; RNA-directed RNA polymerasessuch as the bacteriophage RNA polymerases, also known as RNA replicases;or the bacterial polynucleotide phosphorylases.

[0028] In a preferred embodiment the chain extending enzyme is an RNApolymerase. Said RNA polymerases require the presence of a specificinitiation site within the DNA template. This initiation site, hereinafter referred to as “RNA polymerase promoter sequence”, is the sitewhere the RNA polymerase binds to the DNA template. It is also the siterecognized by the RNA polymerase as an initiation signal, to indicatewhere transcription to form RNA begins.

[0029] Accordingly, the present invention provides oligonucleotidetemplates comprising an RNA polymerase promoter sequence consisting ofdsDNA wherein the polymerase promoter sequence is recognized by an RNApolymerase. The term “recognized” as used herein intends to include alltruncated RNA polymerase promoter sequences shortened by one or morenucleotides at one or either side of the promoter sequence with no orlittle effect on the binding of the RNA polymerase to the initiationsite and with no or little effect on the transcription reaction. Forexample, Milligan et al. (Milligan F. et al., 1987, Nature (15)8783-8798) demonstrated for the T7 RNA polymerase that its promoter doesnot appear to require the DNA in the non-template strand in the region−17 to −14 and −3 to +6, since removing these nucleotides has littleeffect on the transcription reaction. Also, truncation of the templatestrand beyond position +2, i.e. positions +3 to +6, has little effect onthe yield of the reaction (Milligan F. et al., 1987, Nature (15)8783-8798). The thus obtained truncated RNA polymerase promotersequences are meant to be included as “RNA polymerase promoter sequencesrecognized by said RNA polymerase”. Thus, in a specific embodiment ofthe present invention the RNA polymerase promoter sequence consists ofthe truncated RNA polymerase promoter sequence wherein one or morenucleotides are deleted at one or either side of the template strand ofthe promoter sequence. Preferably, the truncated RNA polymerase promoterconsists of the T7 RNA polymerase promoter sequence truncated atpositions +3 to +6 at the 5′end of the template strand as shown in FIG.1.

[0030] Accordingly, it is a first object of the present invention toprovide a method for the synthesis of short double strandedtarget-specific RNAs. The method comprising the steps of a) combining atarget-specific sense oligonucleotide template and a chain extendingenzyme in a reaction mixture such that the template extended senseoligoribonucleotide product is formed; b) combining a target-specificantisense oligonucleotide template and a chain extending enzyme in areaction mixture such that the template extended antisenseoligoribonucleotide product is formed; c) hybridizing the senseoligoribonucleotide product obtained in step a) with the antisenseoligoribonucleotide product obtained in step b).

[0031] The chain-extending enzyme according to the method of theinvention is preferably an RNA polymerase selected from the groupconsisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNApolymerase. In a more preferred embodiment the RNA polymerase consistsof T7 RNA polymerase.

[0032] Accordingly, the oligonucleotide templates used in a methodaccording to the invention, comprise an RNA polymerase promoter sequenceconsisting of dsDNA, wherein the RNA polymerase promoter sequence isrecognized by an RNA polymerase selected from the group consisting of T7RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase. In a preferredembodiment the RNA polymerase promoter sequence is recognized by T7 RNApolymerase. In a more preferred embodiment the T7 RNA polymerasepromoter sequence consists of the truncated T7 RNA polymerase promotersequence as shown in FIG. 1.

[0033] In a further embodiment, the oligonucleotide templates used in amethod according to the invention are characterized by being partiallydouble stranded DNA oligo templates comprising a double stranded RNApolymerase promoter sequence which is extended at the 5′end of thetemplate strand with the target-specific template sequence, optionallyextended with 2 or 3 additional nucleotides. In a more preferredembodiment the target-specific template sequence comprises at the 5′endnucleotides transcribed from the promoter sequence and at the 3′endnucleotides complementary to the nucleotides from the promoter sequence.In the specific embodiment where the oligonucleotide templates comprisethe truncated T7 RNA polymerase promoter sequence shown if FIG. 1, thetarget-specific template sequence comprises at the 5′end two guanosine(g) nucleotides and at the 3′end two cytosine (c) nucleotides.

[0034] Accordingly, it is a second embodiment of the present inventionto provide a method for the synthesis of small interfering RNAs (siRNAs)comprising the steps of a) combining a sense siRNA template with a chainextending enzyme in a reaction mixture such that the template extendedsense oligoribonucleotide product is formed; b) combining an antisensesiRNA template with a chain extending enzyme in a reaction mixture suchthat the template extended antisense oligoribonucleotide product isformed; and c) hybridizing the sense oligoribonucleotide productobtained in step a) with the antisense oligoribonucleotide productobtained in step b); whereby the siRNA templates of step a) and b)comprise a double stranded RNA polymerase promoter sequence extended atthe 5′-end of the template strand with the target-specific templatesequence and 2 or 3 additional nucleotides. In a preferred embodimentthe chain-extending enzyme used in the synthesis of siRNAs consists ofan RNA polymerase, preferably selected from T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. Accordingly the siRNA templates usedin the method according to the invention comprise an RNA polymerasepromoter sequence, which is recognized by an RNA polymerase, selectedfrom the group consisting of T7 RNA polymerase, T3 RNA polymerase andSP6 RNA polymerase. In a more preferred embodiment the chain-extendingenzyme is T7 RNA polymerase. Accordingly, in a preferred embodiment theRNA polymerase promoter sequence of the siRNA templates is recognized byT7 RNA polymerase. In a further embodiment the siRNA template comprisesthe double stranded truncated T7 RNA polymerase promoter sequence asshown in FIG. 1, wherein said truncated T7 RNA polymerase promotersequence is extended at the 5′end of the template strand according tothe method of the invention and wherein the target-specific templatesequence comprises at the 5′end the nucleotides transcribed from thepromoter sequence and at the 3′end nucleotides complementary to thenucleotides transcribed from the promoter sequence. In a specificembodiment the siRNA templates used in a method of the invention,comprise the double stranded truncated T7 RNA polymerase promotersequence as shown in FIG. 1, wherein said truncated T7 RNA polymerasepromoter sequence is extended at the 5′end of the template strandaccording to the method of the invention and wherein the target-specifictemplate sequence comprises at the 5′end two guanosine (g) nucleotidesand at the 3′end two cytosine (c) nucleotides.

[0035] The reaction conditions in either of the aforementioned methodsto obtain a template extended oligoribonucleotide product are generallyknown in the art. In essence, the starting materials for enzymatictranscription to produce RNA are a DNA template, an RNA polymeraseenzyme and the nucleoside triphosphates (NTPs) for the four requiredribonucleotide bases, adenine, cytosine, guanine and uracyl, in areaction buffer optimal for the RNA polymerase enzyme activity. Forexample, the reaction mixture for an in vitro transcription using T7 RNApolymerase typically contains, T7 RNA polymerase (0.05 mg/ml),oligonucleotide templates (1 μM), each NTP (4 mM), and MgCl₂ (25 mM),which supplies Mg²⁺, a co-factor for the polymerase. This mixture isincubated at 37° C. and pH 8.1 (in for example 10 mM Tris-HCl buffer)for several hours (Milligan J. & Uhlenbeck O., 1989, Methods Enzymol(180) 51-62). Kits comprising the aforementioned components arecommercially available such as the MEGA shortscript™ T7 kit (Ambion).

[0036] Purification protocols to obtain the oligoribonucleotide productsfrom either of the above mentioned methods are generally known in theart and comprise amongst others gel electrophoresis, size exclusionchromatography, capillary electrophoresis and HPLC. Gel electrophoreseis typically used to purify the full-length transcripts from thereaction mixture, but this technique is not amendable to production atlarger scale. In a preferred embodiment of the present invention thepurification means to obtain the oligoribonucleotide products consistsof size exclusion chromatography, such as Sephadex G-25 resin,optionally combined with a phenol:chloroform:isoamyl extraction andethanol precipitation.

[0037] It is a third object of the present invention to provide kits toperform the methods according to the invention. In one embodiment thekit comprises one or more of the following components a) instructions todesign target-specific sense and antisense oligonucleotide templates; b)a chain extending enzyme; c) transcriptionbuffers; d) the nucleosidetriphosphates (NTPs) for the four required ribonucleotide bases; e)purification means to obtain the sense and antisense oligoribonucleotideproducts. In a preferred embodiment of the present invention thechain-extending enzyme provided in the kit consists of an RNApolymerase, preferably an RNA polymerase selected from the groupconsisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNApolymerase. Even more preferably the chain extending enzyme provided ina kit according to the invention consist of T7 RNA polymerase.

[0038] The separating means provided in a kit according to the inventiongenerally refers to purification protocols known in the art to obtainoligoribonucleotide products from a reaction mixture and compriseamongst others gel electrophoresis, size exclusion chromatography,capillary electrophoresis and HPLC. In a preferred embodiment of thepresent invention the purification means provided in a kit according tothe invention consists of size exclusion chromatography columns orresins, such as Sephadex G-25 resin.

[0039] The instructions to design target-specific sense and antisenseoligonucleotide templates should contemplate the method exemplified inFIG. 1 of the present invention. In essence the method comprises thefollowing steps;

[0040] 1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-xx(n₁₂₋₃₀)yy-3′. Wherein, x refers to the nucleotides transcribedfrom the promoter, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₂₋₃₀ refers toany oligonucleotide of 12 to 30 nucleotides

[0041] 2) design a sense oligonucleotide template comprising the doublestranded RNA polymerase promoter sequence according to the inventionextended at the 5′end of the template strand with the complementoligonucleotide sequence of the target-specific sequence located in step1), optionally extended with two additional nucleotides.

[0042] 3) design an antisense oligonucleotide template comprising thedouble stranded RNA polymerase promoter sequence according to theinvention extended at the 5′end of the template strand with the reverseoligonucleotide sequence of the target-specific sequence located in step1), optionally extended with two additional nucleotides.

[0043] In a preferred embodiment the methods of the present inventionuse T7 RNA polymerase as chain extending enzyme. In said embodiment themethod to design target-specific sense and antisense oligonucleotidetemplates would comprise the following steps;

[0044] 1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-gg(n₁₂₋₃₀)cc-3′;

[0045] 2) design a sense oligonucleotide template having the followingsequence 5′ TAATACGACTCACTATAGG 3′ ATTATGCTGAGTGATATcc (ncomplement)₁₂₋₃₀ gg—optionally extended with two additional nucleotides,wherein (n complement)₁₂₋₃₀ refers to the complement oligonucleotidesequence of the target-specific sequence located in step 1); and

[0046] 3) design an antisense oligonucleotide template having thefollowing sequence 5′ TAATACGACTCACTATAGG 3′ ATTATGCTGAGTGATATcc (nreverse)₁₂₋₃₀ gg—optionally extended with two additional nucleotides,wherein (n reverse)₁₂₋₃₀ refers to the reverse oligonucleotide sequenceof the target-specific sequence located in step 1).

[0047] In a specific embodiment the methods of the present invention areused for the synthesis of small interfering RNAs (siRNAs). In saidembodiment the method to design target-specific sense and antisensesiRNA templates would comprise the following steps;

[0048] 1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-xx(n₁₅₋₃₀)yy-3′. Wherein, x refers to the nucleotides transcribedfrom the promoter, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₅₋₃₀ refers toany oligonucleotide of 15 to 30 nucleotides;

[0049] 2) design a sense oligonucleotide siRNA template comprising thedouble stranded RNA polymerase promoter sequence according to theinvention extended at the 5′end of the template strand with thecomplement oligonucleotide sequence of the target-specific sequencelocated in step 1), extended with two additional nucleotides, preferablytwo adenine residues;

[0050] 3) design an antisense oligonucleotide siRNA template comprisingthe double stranded RNA polymerase promoter sequence according to theinvention extended at the 5′end of the template strand with the reverseoligonucleotide sequence of the target-specific sequence located in step1), extended with two additional nucleotides, preferably two adenineresidues.

[0051] In the specific embodiment, where the methods to synthesizesiRNAs make use of T7 RNA polymerase as chain extending enzyme, themethod to design target-specific sense and antisense siRNA templateswould comprise the following steps;

[0052] 1) look for a target-specific sequence located within the codingsequence of the target gene and having the following sequence5′-gg(n₁₅₋₃₀)cc-3′;

[0053] 2) design a sense oligonucleotide siRNA template having thefollowing sequence 5′ TAATACGACTCACTATAGG 3′ ATTATGCTGAGTGATATcc (ncomplement)₁₅₋₃₀ gg aa

[0054] wherein (n complement)₁₅₋₃₀ refers to the complementoligonucleotide sequence of the target-specific sequence located in step1); and

[0055] 3) design an antisense oligonucleotide siRNA template having thefollowing sequence 5′ TAATACGACTCACTATAGG 3′ ATTATGCTGAGTGATATcc (nreverse)₁₅₋₃₀ gg aa

[0056] wherein (n reverse)₁₅₋₃₀ refers to the reverse oligonucleotidesequence of the target-specific sequence located in step 1).

[0057] Accordingly, the present invention provides a kit for thesynthesis of short double stranded target-specific RNAs the kitcomprising at least one of the following components; a) instructions todesign target-specific sense and antisense oligonucleotide templates; b)a chain extending enzyme; c) transcriptionbuffers; d) the nucleosidetriphosphates (NTPs) for the four required ribonucleotide bases; e)purification means to obtain the sense and antisense oligoribonucleotideproducts.

[0058] Thus in a further embodiment the present invention provides kitsfor the synthesis of small interfering RNAs the kit comprising at leastone of the following components; a) instructions to designtarget-specific sense and antisense siRNA templates; b) a chainextending enzyme; c) transcriptionbuffers; d) the nucleosidetriphosphates (NTPs) for the four required ribonucleotide bases; e)purification means to obtain the sense and antisense oligoribonucleotideproducts.

[0059] It is also an object of the present invention to provide themeans for any of the disclosed methods for the in vitro synthesis ofshort double stranded RNAs. Accordingly the present invention provides;

[0060] i) a method to design target-specific sense and antisenseoligonucleotide templates

[0061] ii) a chain extending enzyme according to the invention for usein a method for the in vitro synthesis of short double stranded RNAs

[0062] iii) purification means to obtain the sense and antisenseoligoribonucleotide products.

[0063] iv) reagents for the reaction mixture such that the sense andantisense oligoribonucleotide products are formed from thetarget-specific sense and antisense oligonucleotide templates using achain extending enzyme according to the invention

[0064] It is a further object of the present invention to use the siRNAsobtainable by a method of the present invention in a process forinhibiting expression of a target gene in a cell. The process comprisingintroduction of siRNAs obtainable by a method of the present invention,into a cell.

[0065] The target gene may be a gene derived from the cell (i.e., acellular gene), an endogenous gene (i.e., a cellular gene present in thegenome), a transgene (i.e., a gene construct inserted at an ectopic sitein the genome of the cell), or a gene from a pathogen which is capableof infecting an organism from which the cell is derived. Depending onthe particular target gene and the dose of double stranded RNA materialdelivered, this process may provide partial or complete loss of functionfor the target gene.

[0066] The cell with the target gene may be derived from or contained inany organism. The organism may a plant, animal, protozoan, bacterium,virus, or fungus. The plant may be a monocot, dicot or gymnosperm; theanimal may be a vertebrate or invertebrate. The cell having the targetgene may be from the germ line or somatic, totipotent or pluripotent,dividing or non-dividing, parenchyma or epithelium, immortalized ortrans- formed, or the like. The cell may be a stem cell or adifferentiated cell. Cell types that are differentiated includeadipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons,glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

[0067] The isolated RNA obtainable by a method of the present inventionconsists of target-specific short double stranded RNAs, wherein saidtarget-specific short double stranded RNAs are less than 50 nucleotides,preferably less than 30 nucleotides long, even more preferably 30-12nucleotides, characterized by comprising at the 5′end nucleotidestranscribed from the promoter sequence and at the 3′end nucleotidescomplementary to the nucleotides transcribed from the promoter sequence,preferably the number of nucleotides transcribed from the promotersequence and the number of nucleotides complementary to the nucleotidestranscribed from the promoter sequence consist of 2, 3, or 4nucleotides, more preferably of 2 nucleotides.

[0068] The short double stranded RNAs obtainable by a method of thepresent invention are optionally extended at the 3′end with 2 or 3additional nucleotides and could in a further embodiment of the presentinvention being represented as having the following sense sequence5′-xx(n₁₂₋₃₀)yy-3′ wherein x refers to the nucleotides transcribed fromthe promoter sequence, y refers to the nucleotides complementary to thenucleotides transcribed form the promoter sequence, and n₁₂₋₃₀ refers toany oligonucleotide of 12 to 30 nucleotides. In a specific embodimentthe short double stranded RNAs have as sense sequence 5′-gg(n₁₅₋₃₀)cc-3′wherein g refers to the nucleotide guanosine transcribed from thetruncated T7 RNA polymerase promoter sequence (as shown in FIG. 1), crefers to the nucleotide cytosine complementary to the nucleotidestranscribed form the truncated T7 RNA polymerase promoter (as shown inFIG. 1) sequence, and n₁₅₋₃₀ refers to any oligonucleotide of 15 to 30nucleotides.

[0069] The RNA may be directly introduced into the cell (i.e.,intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing an organism in a solutioncontaining the RNA. Methods for oral introduction include direct mixingof the RNA with food of the organism, as well as engineered approachesin which a species that is used as food is engineered to express theRNA, then fed to the organism to be affected. For example, the RNA maybe sprayed onto a plant or a plant may be genetically engineered toexpress the RNA in an amount sufficient to kill some or all of apathogen known to infect the plant.

[0070] Physical methods of introducing nucleic acids, for example,injection directly into the cell or extracellular injection into theorganism, may also be used. We disclose herein that in HeLa cells,double-stranded RNA introduced outside the cell inhibits geneexpression.

[0071] Vascular or extravascular circulation, the blood or lymph system,the phloem, the roots, and the cerebrospinal fluid are sites where theRNA may be introduced. A transgenic organism that expresses RNA from arecombinant construct may be produced by introducing the construct intoa zygote, an embryonic stem cell, or another multipotent cell derivedfrom the appropriate organism.

[0072] Physical methods of introducing nucleic acids include injectionof a solution containing the RNA, bombardment by particles covered bythe RNA, soaking the cell or organism in a solution of the RNA orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, promote annealing of the duplex strands,stabilize the annealed strands, or otherwise increase inhibition of thetarget gene.

[0073] The present invention may be used to introduce RNA into a cellfor the treatment or prevention of disease. For example, dsRNA may beintroduced into a cancerous cell or tumor and thereby inhibit geneexpression of a gene required for maintenance of thecarcinogenic/tumorigenic phenotype. To prevent a disease or otherpathology, a target gene may be selected which is required forinitiation or maintenance of the disease/pathology. Accordingly, in afurther embodiment the invention provides a pharmaceutical compositioncomprising short double stranded RNAs obtainable by a method of thepresent invention to inhibit gene expression of a target gene and anappropriate carrier. The composition may be administered in any suitableway, e.g. by injection, by oral, intra-ocular, topical, nasal, rectalapplication etc. The carrier may by any suitable pharmaceutical carrier,preferably, a carrier is used, which is capable of increasing theefficacy of the RNA molecules to enter the target cells, for exampleliposomes, natural viral capsids or by chemically or enzymaticallyproduced artificial capsids or structures derived therefrom.

[0074] Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use ofdouble-stranded RNA to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics would envision determining the function of uncharacterizedgenes by employing the invention to reduce the amount and/or alter thetiming of target gene activity. The invention could be used indetermining potential targets for pharmaceutics, understanding normaland pathological events associated with development, determiningsignaling pathways responsible for postnatal development/aging, and thelike. The increasing speed of acquiring nucleo-tide sequence informationfrom genomic and expressed gene sources, including total sequences forthe human, mouse, yeast, D. melanogaster, and C. elegans genomes, can becoupled with the invention to determine gene function in an organism(e.g., nematode). The preference of different organisms to useparticular codons, searching sequence databases for related geneproducts, correlating the linkage map of genetic traits with thephysical map from which the nucleotide sequences are derived, andartificial intelligence methods may be used to define putative openreading frames from the nucleotide sequences acquired in such sequencingprojects.

[0075] A simple assay would be to inhibit gene expression according tothe partial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product.

[0076] It is thus an object of the present invention to provide a methodto inhibit expression of a target gene in a cell comprising introductionof RNA into a cell wherein said RNA comprises target-specific shortdouble stranded RNA, wherein said target-specific short double strandedRNA is less than 50 nucleotides, preferably less than 30 nucleotideslong, even more preferably 30-12 nucleotides long, characterized bycomprising at the 5′end nucleotides transcribed from the promotersequence and at the 3′end nucleotides complementary to the nucleotidestranscribed from the promoter sequence wherein said promoter sequence isbeing recognized by an RNA polymerase. In a further embodiment thepromoter sequence is being recognized by an RNA polymerase selected fromthe group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 RNApolymerase.

[0077] This invention will be better understood by reference to theExperimental Details that follow, but those skilled in the art willreadily appreciate that these are only illustrative of the invention asdescribed more fully in the claims that follow thereafter. Additionally,throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

EXAMPLE 1 EGFP and GL3 Specific Short dsRNAs Transcribed In Vitro,Induce RNA Interference in Human Cells

[0078] Materials and Methods

[0079] Plasmid Constructs

[0080] Luciferase+ was expressed from the plasmid pGL3-control(Promega). EGFP was expressed from EGFP/pcDNA5-FRT, which contains theEGFP gene from pEGFP (Clontech) directionally ligated into the HindIIIand NotI sites of the mammalian expression vector pcDNA5/FRT(Invitrogen).

[0081] In Vitro Transcription and Hybridization of siRNAs

[0082] Oligo template strands were hybridized to a sense T7 promotersequence (5′TAATACGACTCACTATAGG) in 10 mM Tris-HCl pH 9.0, 100 mM NaCl,1 mM EDTA by boiling for 2′ and cooling slowly to room temperature over2-3 hr. Transcription was performed using the MEGAshortscript™ T7 kit(Ambion) according to the manufacturer's instructions. siRNA strandswere purified over G-25 spin columns, phenol:chloroform:isoamyl alcohol(25:24:1) extracted using Heavy Phase-Lock Gels (Eppendorf), and ethanolprecipitated overnight at −80° C. Complementary siRNA strands werehybridized in 1 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0 by boiling for 2′and cooling slowly to room temperature over 2-3 hr. Hybridization wasassessed by running the ds- and ss-siRNAs on non-denaturing 20%polyacrylamide TBE gels.

[0083] Cell Lines and Transfection

[0084] HeLa cells were grown in DMEM with high glucose and 1-glutamine(Invitrogen) supplemented with 1.8 mM 1-glutamine, 9% FBS, and 45 U/lpen/strep. Cells were transfected in a manner similar to that describedin Elbashir et al. (2001). 24 hr before transfection, cells weretrypsinized and diluted with growth medium lacking antibiotics to 3×10⁵cells/ml. 0.5 ml of cells were seeded into each well of a 24-well plate.The cells were transfected with 1 μg of GL3-control or EGFP/pcDNA5-FRTreporter constructs and 50 pmol of single-stranded or 25 pmoldouble-stranded siRNAs, except where otherwise noted, usingLipofectamine™ 2000 (LF2000; Invitrogen) according to the manufacturer'sinstructions. Specifically, we used 21 μl of LF2000 per well in 48 μl ofserum-free medium lacking antibiotics. The diluted LF2000 waspre-incubated at room temperature for 1′ prior to mixing with reporterand/or siRNAs diluted in the same medium to 50 μl total volume.Complexes were then incubated at room temperature for 20′ before beingadded to the cells. EGFP and GL3 reporter gene assays were performedafter 24 hrs. For the JNK2α1 siRNA experiment and GL3 siRNA doseresponse experiment, 6-well plates were used. Cell numbers wereincreased 4-fold and reagent amounts 5-fold. For the JNK2α1 siRNAexperiment, cells were harvested for RNA isolation and proteinextraction were approximately 48 hr post-transfection.

[0085] Reporter Gene Assays

[0086] FACS analysis of EGFP-transfected cells was performed using aFACScan (Beckton-Dickinson). Cells were trypsinized and washed with PBSprior to resuspension in FACS fixing solution (PBS+1% formaldehyde).Transfection efficiencies were estimated by comparing samplestransfected with water with those transfected with EGFP/pcDNA5-FRT andwere typically 75-90%. The extent of RNAi induced by transcribed orsynthetic siRNAs was estimated from the change in the mean GFPfluorescence in samples with or without cotransfected siRNAs.

[0087] For luciferase assays, cells were trypsinized and 100 μl aliquotswere transferred to triplicate wells in white 96-well tissue cultureplates. Assays were performed using the Luc-Screen™ System (AppliedBiosystems) and a TopCount-NXT™ Microplate Scintillation andLuminescence Counter (Packard) according to the manufacturers'instructions.

[0088] Northern and Western Blotting

[0089] Total RNA was prepared from samples of approximately 10⁶ HeLacells using the RNeasy Mini Kit (Qiagen) according to the manufacturer'sinstructions. Samples were run on pre-cast MOPS Latitude RNA agarosegels (BioWhittaker Molecular Applications) and transferred to Hybond-XLnylon membranes (AP Biotech) according to the manufacturers'instructions. DNA probes were made using the Rediprime II system (APBiotech) according to the manufacturer's instructions. Hybridization wasperformed in Rapid-Hyb solution (AP Biotech) according to themanufacturer's instructions.

[0090] Nuclear and cytoplasmic protein extracts were made by the methoddescribed in Gordon (1991), substituting Complete Protease InhibitorCocktail (Roche) for leupeptin, aprotinin, and pepstatin and omittingsodium deoxycholate.

[0091] Extracts were run on 4-20% SDS polyacrylamide minigels(Invitrogen) with Rainbow protein marker (AP Biotech) and electroblottedonto 0.2 μm Transblot nitrocellulose membranes (BioRad). The blots wererinsed in PBS+0.05% Tween-20 and incubated overnight at 4° C. with 1:150diluted JNK2 D2 mouse monoclonal antibody (Santa Cruz Biotech) inPBS/Tween-20 with 5% milk powder. The blots were then washed three timesin PBS/Tween-20 before a 45 min incubation with 1:3000 HRP-conjugatedgoat anti-mouse antibodies (BioRad). Three more washes and a 30 minincubation in PBS/Tween-20 were performed before detection by the ECLsystem (AP Biotech).

[0092] Results

[0093] RNAi has been previously demonstrated using siRNAs that aredouble-stranded except for two 3′ overhanging nucleotides (Elbashir etal. 2001; Caplen et al. 2001). In order to relatively quickly andinexpensively create a variety of siRNAs for multiple cellular targets,we designed a scheme to generate the molecules using in vitrotranscription techniques.

[0094] Milligan et al. (1987) describe the use of partiallysingle-stranded DNA oligo templates for transcription by T7 DNApolymerase. The standard T7 minimal promoter includes three guanosinenucleotides at the 3′ end which are incorporated as the first threebases of the transcript. However, the third guanosine can usually bereplaced with other nucleotides without significant reduction of invitro transcription yields (Milligan et al. 1987). Therefore, siRNAsproduced by this method should include two 5′ guanosine nucleotides. Twocomplementary cytosine nucleotides are needed near the 3′ end of eachsiRNA strand to base pair with the 5′ guanosines on the other strand.siRNAs of a given length produced in this manner should be able totarget sequences appearing approximate once every 250 nucleotides onaverage in an mRNA.

[0095] We designed DNA oligo templates with the constraints above inmind (FIG. 1). Each template is used to transcribe one strand of ansiRNA. The strands are crudely purified by passage over a Sephadex G-25size exclusion column, phenol:chloroform extraction, and ethanolprecipitation. The strands are then resuspended in annealing buffer andhybridized by boiling and slow cooling.

[0096] Our in vitro transcribed siRNAs differ from the chemicallysynthesized variety used previously in two ways. First, all otherreported siRNAs have been highly purified. Second, in vitro transcribedsiRNAs retain a 5′ triphosphate group. Like the siRNA species producedin vivo as part of the natural RNAi mechanism, chemically synthesizedRNA oligonucleotides used to make siRNAs have carried 5′ monophosphates.In order to determine if these differences render our siRNAs incompetentto induce RNAi, we first tested double-stranded siRNAs designed totarget two reporter genes—EGFP and GL3 (FIGS. 2 and 3). Mean results andstandard errors from at least three independent experiments are shown(FIG. 2B, FIG. 3B).

[0097] The transcribed GL3 ds siRNA 1 reduced luciferase activity fromthe cotransfected pGL3-control reporter plasmid by approximately 5-foldwhile the antisense strand alone in double-molar concentration had noeffect. A similar result was observed with a chemically synthesized GL3ds siRNA (ds1 RNA oligos). The second transcribed siRNA had a moremodest effect. While the third transcribed siRNA had a strong effect,significant activity was also seen from the antisense strand alone. Thestrength of the effect from the double-stranded species is dosedependent (FIG. 2C) and modifies steady-state RNA levels. EGFP ds siRNA2 also had a modest effect on luciferase activity (FIG. 2B). However,this effect appears to be non-specific and shows a limited response atincreasing doses.

[0098] The same transcribed EGFP ds siRNA 2 strongly reduced GFPfluorescence in cells cotransfected with the EGFP/pcDNA5-FRT reporter(FIG. 3B). Much more modest effects were evident from sense or antisensestrands alone or from a chemically synthesized siRNA (EGFP ds1 RNAoligos) or its component parts. GFP fluorescence was not affected by thenon-specific GL3 ds siRNA 3.

[0099] The above mentioned in vitro transcribed (IVT) luciferase dssiRNAs yielded inhibition of luciferase activity to a different extent.As a positive control for RNAi activity, we used a chemicallysynthesized luciferase ds siRNA. In our hands, luciferase activity fromthe cotransfected pGL3-control reporter plasmid was reducedapproximately 5-fold by the synthetic ds siRNAs. Transfectionefficiencies for all experiments varied between 91 and 95%. OneIVT-luciferase ds siRNA (GL3 ds siRNA 1) reduced luciferase activity78%, while the antisense RNA strand alone at twice the molarconcentration of the ds siRNA had no effect. The second IVT-siRNA (GL3ds siRNA 2) we tested had a more modest effect (36% inhibition). Whilethe third IVT-siRNA had a strong effect (82% inhibition), significantactivity was also seen from the antisense RNA strand alone (55%inhibition), confounding the result. A mixture of the three IVT-siRNAs,each at one-third the molar concentration used for them individually,had an intermediate effect (70% inhibition) rather than a synergisticone, suggesting that there may be no advantage to using multiple siRNAsto target the same gene. We could show the inhibitory effect from the dsspecies to be dose-dependent. While a non-specific GFP siRNA (GFP dssiRNA 2) also had a modest effect (46% inhibition) on luciferaseactivity, this appears to be non-specific and shows a limited responseat increasing doses (data not shown).

[0100] The same IVT-GFP ds siRNA (GFP ds siRNA 2) strongly reduced GFPfluorescence in cells cotransfected with the GFP/pcDNA5-FRT reporter(87% inhibition). Much more modest effects were evident from sense (41%)or antisense (19%) strands alone or from a chemically synthesized siRNA(GFP ds1 RNA oligo) or its component parts. GFP fluorescence was, asexpected, not affected by the non-specific luciferase ds siRNA 3.

[0101] To demonstrate that endogenous gene expression can also beaffected by transcribed siRNAs, we targeted the products of the JNK2α1(FIG. 4A) and CDS-1 (FIG. 4B) genes. Western and Northern blot analysisrevealed specific reduction of JNK2α1 protein and RNA levels in samplesin nuclear extracts of HeLa cells transfected with either a transcribedsiRNA (JNK2α1 ds siRNA 1—estimated 87% reduction) or a chemicallysynthesized siRNA (JNK2α1 ds1 RNA oligos—estimated 76% reduction) whencompared to cells transfected with water (mock), EGFP/pcDNA5-FRT plasmidas a transfection control (EGFP DNA only), single strands of siRNAs, ora non-specific siRNA (EGFP ds siRNA 2).

[0102] Western blot analysis revealed modest (up to 67%) reduction ofCDS 1 protein levels in cytoplasmatic extracts of HeLa cells transfectedwith CDS 1-specific IVT-siRNAs (FIG. 4) but not in cells transfectedwith an unspecific siRNA when compared to mock-transfected cells.

EXAMPLE 2 Mouse Insr Specific Short dsRNAs Transcribed In Vitro,Knockdown Insr in Liver of Balb/C Mice

[0103] Male Balb/C mice (approx 25 g) (standard housing, free access tochow/water) received a tail vein injection of either saline, 2.3 ml, orsaline containing 40 micrograms of siRNA directed against the murineinsulin receptor (NCBI accession number NM_(—)010568; bases 2536-2556)prepared,by the truncated T7 promoter method of in vitro transcription,along with 800 U RNase inhibitor.

[0104] The injections were administered as rapidly as possible (8-10seconds). Two control and two siRNA treated mice were sacrificed at 24,at 48 and at 72 hours; the liver was quickly removed,weighed, and frozenin dry ice/isopropanol. Total RNA was extracted using pulverized frozentissue and RNEasy Maxi kits (Qiagen).

[0105] After first strand cDNA synthesis, mRNA for the insulin receptorwas assayed by Q-PCR using the Smart Cycler (primers: F 3526-3548, R3744-3768) and results were normalized to cyclophilin A expression alsoby Q-PCR (bases 157-182 and 496-521 of NCBI accession numberNM_(—)017101).

1 18 1 19 RNA Artificial Sequence 19nt within the coding sequence of theJNK2alpha1 mRNA 1 ggaucaugaa agaaugucc 19 2 19 DNA Artificial Sequence5′ strand of the sense oligonucleotide template for T7 RNA polymerasetranscription 2 taatacgact cactatagg 19 3 38 DNA Artificial Sequence 3′strand of the sense oligonucleotide template for T7 RNA polymerasetranscription 3 attatgctga gtgatatcct agtactttct tacaggaa 38 4 19 DNAArtificial Sequence 5′ strand of the antisense oligonucleotide templatefor T7 RNA polymerase transcription 4 taatacgact cactatagg 19 5 38 DNAArtificial Sequence 3′ strand of the antisense oligonucleotide templatefor T7 RNA polymerase transcription 5 attatgctga gtgatatcct gtaagaaagtactaggaa 38 6 19 RNA Artificial Sequence 19 nt target sequence withinthe coding sequence of GL3 6 ggcuaugaag agauacgcc 19 7 19 RNA ArtificialSequence 19 nt target sequence within the coding sequence of GL3 7gggcauuucg cagccuacc 19 8 19 RNA Artificial Sequence 19 nt targetsequence within the coding sequence of GL3 8 ggugccaacc cuauucucc 19 919 RNA Artificial Sequence 19 nt target sequence within the codingsequence of GL3 9 cuuacgcuga guacuucga 19 10 19 RNA Artificial Sequence19 nt target sequence within the coding sequence of EGFP 10 gggcgaggagcuguucacc 19 11 19 RNA Artificial Sequence 19 nt target sequence withinthe coding sequence of EGFP 11 ccaccggcaa gcugcccgu 19 12 19 RNAArtificial Sequence 19 nt target sequence within the coding sequence ofJNK2alpha1 12 ggaucaugaa agaaugucc 19 13 20 RNA Artificial Sequence 20nt target sequence within the coding sequence of JNK2alpha1 13gguguuguaa aagaucagcc 20 14 19 RNA Artificial Sequence 19 nt targetsequence within the coding sequence of JNK2alpha1 14 ccaagggauuguuugugcu 19 15 19 RNA Artificial Sequence 19 nt target sequence withinthe coding sequence of CDS1 15 ggcagcguua cccaguccc 19 16 19 RNAArtificial Sequence 19 nt target sequence within the coding sequence ofCDS1 16 ggcuccuccu cacaguccc 19 17 19 RNA Artificial Sequence 19 nttarget sequence within the coding sequence of CDS1 17 ggagccuaccccugccccc 19 18 19 RNA Artificial Sequence 19 nt target sequence withinthe coding sequence of CDS1 18 ggaaaggaaa acgccgucc 19

1. A method for the synthesis of target-specific short double stranded RNAs comprising the steps of: a) combining a target-specific sense oligonucleotide template and a chain extending enzyme in a reaction mixture such that the template extended, sense oligoribonucleotide, product is formed; b) combining a target specific antisense oligonucleotide template and a chain extending enzyme in a reaction mixture such that the template extended, antisense oligoribonucleotide, product is formed; and c) hybridizing the sense oligoribonucleotide product obtained in step a) with the complementary antisense oligoribonucleotide product obtained in step b).
 2. A method according to claim 1 wherein the chain extending enzyme is an RNA polymerase.
 3. A method according to claim 2 wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase of and SP6 polymerase.
 4. A method according to claim 1 wherein the oligonucleotide templates of step a) and b) comprise an RNA polymerase promoter sequence consisting of dsDNA.
 5. A method according to claim 4 wherein the RNA polymerase promoter sequence is recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.
 6. A method according to claim 4 wherein the RNA polymerase promoter sequence is recognized by T7 RNA polymerase.
 7. (Currently Amended) A method according to any one of claims 4 to 6 claim 4 wherein the oligonucleotide templates of step a) and b) are further characterized by being partially double stranded DNA oligo templates comprising a double stranded RNA polymerase promoter sequence which is extended at the 5′end of the template strand with the target specific template sequence.
 8. A method according to claim 4 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG.
 1. 9. A method according to claim 1 wherein the target-specific short double stranded RNA is less than 30 nucleotides long.
 10. A method according to claim 1 wherein the target-specific short double stranded RNA is characterized by comprising at the 5′-end nucleotides transcribed from the promoter sequence and at the 3′-end nucleotides complementary to the nucleotides transcribed from the promoter sequence.
 11. A method for the synthesis of small interfering RNAs (siRNAs) comprising the steps of; a) combining a sense siRNA template with a chain extending enzyme in a reaction mixture such that the template extended sense oligoribonucleotide product is formed; b) combining an antisense siRNA template with a chain extending enzyme in a reaction mixture such that the template extended antisense oligoribonucleotide product is formed; and c) hybridizing the sense oligoribonucleotide product obtained in step a) with the antisense oligoribonucleotide product obtained in step b); whereby the siRNA templates of step a) and b) comprise a double stranded RNA polymerase promoter sequence extended at the 5′-end of the template strand with the target-specific template sequence and 2 or 3 additional nucleotides.
 12. A method according to claim 11 wherein the chain-extending enzyme is an RNA polymerase.
 13. A method according to claim 12 wherein the RNA polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.
 14. A method according to claim 12 wherein the RNA polymerase consists of T7 RNA polymerase.
 15. A method according to claim 11 wherein the RNA polymerase promoter sequence is recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.
 16. A method according to claim 15 wherein the RNA polymerase promoter sequence is recognized by T7 RNA polymerase.
 17. A method according to claim 16 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG.
 1. 18. A method according to claim 11 wherein the target-specific template sequence is further characterized by comprising at the 5′end two guanosine (g) nucleotides and at the 3′end two cytosine (c) nucleotides.
 19. A method to inhibit expression of a target gene in a cell comprising introduction of RNA into a cell wherein said RNA comprises target-specific short double stranded RNA of less than 50 nucleotides characterized by comprising at the 5′end nucleotides transcribed from an RNA polymerase promotor sequence and at the 3′end nucleotides complementary to the nucleotides transcribed from said RNA polymerase promotor sequence.
 20. A method according to claim 19 wherein the number of nucleotides transcribed from the promoter sequence and the number of nucleotides complementary to the nucleotides transcribed from the promoter sequence consist of 2, 3 or 4 nucleotides.
 21. A method according to claim 19 wherein the target-specific short double stranded RNA is extended at the 3′ends with 2 or 3 additional nucleotides.
 22. A method according to claim 19 wherein the RNA polymerase promoter sequence is being recognized by an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase or SP6 polymerase.
 23. A method according to claim 19 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG.
 1. 24. A method according to claim 19 wherein the target gene is a cellular gene, an endogenous gene, a transgene, or a gene from a pathogen.
 25. Isolated RNA obtainable by the method of claim
 1. 26. Isolated RNA according to claim 25 wherein said RNA consists of target-specific short double stranded RNA of less than 50 nucleotides, characterized by comprising at the 5′end nucleotides transcribed from an RNA polymerase promoter sequence and at the 3′end nucleotides complementary to the nucleotides transcribed from said RNA polymerase promoter sequence.
 27. Isolated RNA according to claim 25 wherein the number of nucleotides transcribed from the promoter sequence and the number of nucleotides complementary to the nucleotides transcribed from the promoter sequence consist of 2, 3 or 4 nucleotides.
 28. Isolated RNA according to claim 25 wherein the sense sequence of said RNA molecule can be represented by 5′-xx(n₁₂₋₃₀)yy-3′ wherein x refers to the nucleotides transcribed from the promoter sequence, y refers to the nucleotides complementary to the nucleotides transcribed from the promoter sequence, and n refers to any oligonucleotide of 12 to 30 nucleotides.
 29. Isolated RNA according to claim 25 wherein the sense sequence of said RNA molecule can be represented by 5′-gg(n₁₅₋₃₀)cc-3′ wherein g refers to the nucleotide guanosine transcribed from the truncated T7 RNA polymerase promoter sequence (as shown in FIG. 1), c refers to the nucleotide cytosine complementary to the nucleotides transcribed form the truncated T7 RNA polymerase promoter (as shown in FIG. 1) sequence, and n₁₅₋₃₀ refers to any oligonucleotide of 15 to 30 nucleotides.
 30. Isolated RNA according to claim 25 wherein the target-specific short double stranded RNA is extended at the 3 ′ ends with 2 or 3 additional nucleotides.
 31. A target-specific sense oligonucleotide template for use in a method according claim
 18. 32. A target-specific antisense oligonucleotide template for use in a method according to claim
 1. 33. The target-specific oligonucleotide template of claim 31 wherein said oligonucleotide templates comprise an RNA polymerase promoter sequence consisting of dsRNA which is extended at the 5′end of the template strand with the target-specific template sequence.
 34. The target-specific oligonucleotide templates of claim 31 wherein the RNA polymerase promoter sequence consists of the truncated T7 RNA polymerase promoter sequence as shown in FIG.
 1. 35. The target-specific oligonucleotide templates of claim 31 wherein the target-specific short double stranded RNA is less than 30 nucleotides long.
 36. The target-specific oligonucleotide templates of claim 31 wherein the target-specific short double stranded RNA is characterized by comprising at the 5′-end nucleotides transcribed from the promoter sequence and at the 3′-end nucleotides complementary to the nucleotides transcribed from the promoter sequence.
 37. A kit for the synthesis of short double stranded target-specific RNAs the kit comprising at least one of the following components; a) instructions to design target-specific sense and antisense oligonucleotide templates; b) a chain extending enzyme; c) transcriptionbuffers; d) the nucleoside triphosphates (NTPs) for the four required ribonucleotide bases; e) purification means to obtain the sense and antisense oligoribonucleotide products.
 38. A kit according to claim 37 wherein the instructions to design the target-specific sense and antisense oligonucleotide templates consists of the method comprising the following steps; 1) look for a target-specific sequence located within the coding sequence of the target gene and having the following sequence 5′-xx(n₁₂₋₃₀)yy-3′. Wherein, x refers to the nucleotides transcribed from the promoter, y refers to the nucleotides complementary to the nucleotides transcribed form the promoter sequence, and n₁₂₋₃₀ refers to any oligonucleotide of 12 to 30 nucleotides 2) design a sense oligonucleotide template comprising the double stranded RNA polymerase promoter sequence according to the invention extended at the 5′end of the template strand with the complement oligonucleotide sequence of the target-specific sequence located in step 1), optionally extended with two additional nucleotides. 3) design an antisense oligonucleotide template comprising the double stranded RNA polymerase promoter sequence according to the invention extended at the 5′end of the template strand with the reverse oligonucleotide sequence of the target-specific sequence located in step 1), optionally extended with two additional nucleotides.
 39. A kit according to claim 37 wherein the chain extending enzyme consists of an RNA polymerase, preferably an RNA polymerase selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
 40. A kit according to claim 37 wherein the purification means consist of size exclusion chromatography columns. 